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Hydrocortisone (HYD) is one of the corticosteroids that illegally used in cosmetic products, which could lead to a potential health problem. In this work, HYD was conjugated to bovine serum albumin via a succinic anhydride spacer arm to yield an immunogen. New Zealand rabbits were immunized to obtain a polyclonal antibody against HYD. Based on the pAb, a heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) by using progesterone carboxylic acid as a competitive hapten was developed. The assay showed an IC50 of 0.50 ng mL−1, detection limit of 0.04 ng mL−1 and a linear range of 0.10–2.00 ng mL−1. The assay was applied to determine HYD in cosmetic samples. Recoveries from 82.5% to 118.9% and CV from 1.3% to 9.5% were obtained using a simple pretreatment. Good correlation between the results of ciELISA and standard LC–MS/MS was obtained, suggesting its potential usage in HYD monitoring in cosmetic samples. [ABSTRACT FROM AUTHOR]